Functional identification of genes causing estrogen independence of human breast cancer cells Ton Agthoven, Jos Veldscholte, Marcel Smid, Thecla L. A. Agthoven, Lilian Vreede, Marieke Broertjes, Ingrid Vries, Danielle Jong, Roya Sarwari, Lambert C. J. Dorssers To cite this version: Ton Agthoven, Jos Veldscholte, Marcel Smid, Thecla L. A. Agthoven, Lilian Vreede, et al.. Functional identification of genes causing estrogen independence of human breast cancer cells. Breast Cancer Research and Treatment, Springer Verlag, 2008, 114 (1), pp.23-30. 10.1007/s10549-008-9969-5. hal- 00478328 HAL Id: hal-00478328 https://hal.archives-ouvertes.fr/hal-00478328 Submitted on 30 Apr 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Breast Cancer Res Treat (2009) 114:23–30 DOI 10.1007/s10549-008-9969-5 PRECLINICAL STUDY Functional identification of genes causing estrogen independence of human breast cancer cells Ton van Agthoven Æ Jos Veldscholte Æ Marcel Smid Æ Thecla L. A. van Agthoven Æ Lilian Vreede Æ Marieke Broertjes Æ Ingrid de Vries Æ Danielle de Jong Æ Roya Sarwari Æ Lambert C. J. Dorssers Received: 5 March 2008 / Accepted: 5 March 2008 / Published online: 21 March 2008 Ó Springer Science+Business Media, LLC. 2008 Abstract Endocrine treatment of breast cancer is widely mechanisms of breast tumor progression and therapy resis- applied and effective. However, in advanced disease cases, tance thereby offering novel targets for the development of the tumors will eventually progress into an estrogen-inde- tailor-made therapeutical and prevention strategies. pendent and therapy-resistant phenotype. To elucidate the molecular mechanisms underlying this endocrine therapy Keywords Cell proliferation control Á Functional screen Á failure, we applied retroviral insertion mutagenesis to iden- Genetic screen Á Insertion mutagenesis Á tify the main genes conferring estrogen independence to Endocrine therapy Á Tamoxifen resistance human breast cancer cells. Estrogen-dependent ZR-75-1 cells were infected with replication-defective retroviruses followed by selection with the anti-estrogen 4-hydroxy- tamoxifen. In the resulting panel of 79 tamoxifen-resistant Introduction cell lines, the viral integrations were mapped within the human genome. Genes located in the immediate proximity of Endocrine therapy of breast cancer has been applied widely the retroviral integration sites were characterized for altered and has proven to be effective. Breast tumor development expression and their capacity to confer anti-estrogen resis- and recurrence can be reduced by long-term anti-estrogen tance when transfected into breast cancer cells. Out of 15 treatment with tamoxifen (or novel selective estrogen candidate BCAR (breast cancer anti-estrogen resistance) receptor modulators) and aromatase inhibitors [1]. In genes, seven (AKT1, AKT2, BCAR1, BCAR3, EGFR, GRB7, addition, tamoxifen and other endocrine treatment regi- and TRERF1/BCAR2) were shown to directly underlie mens have shown clinical benefit in advanced disease [2]. estrogen independence. Our results show that insertion Tamoxifen competes with estrogen for the estrogen mutagenesis is a powerful tool to identify BCAR loci, which receptor (ER) a and interferes effectively with estrogen may provide insights into the molecular and cellular signaling. However, in many instances in advanced dis- ease, endocrine treatments ultimately fail due to the development of therapy resistance. Notwithstanding the Ton van Agthoven and Jos Veldscholte contributed equally to this fact that estrogen antagonists and aromatase inhibitors research. exhibit alternative modes of action, in nearly all cases Electronic supplementary material The online version of this tumor cells eventually become estrogen-independent for article (doi:10.1007/s10549-008-9969-5) contains supplementary growth. Despite the detailed insights in ER function, the material, which is available to authorized users. mechanism of this general therapy failure is still poorly T. van Agthoven Á J. Veldscholte Á M. Smid Á understood [3–7]. Gene expression profiling has become a T. L. A. van Agthoven Á L. Vreede Á M. Broertjes Á I. de Vries Á powerful tool to identify gene sets associated with disease D. de Jong Á R. Sarwari Á L. C. J. Dorssers (&) progression and clinical outcome [8–11]. However, only a Department of Pathology, Josephine Nefkens Institute, Be 432, minority of these genes hint at the underlying mechanisms, Erasmus MC-University Medical Center Rotterdam, P.O. Box 2040, Rotterdam, CA 3000, The Netherlands and most just represent markers. Here we have undertaken e-mail: [email protected] an extensive study into the mechanisms leading to estrogen 123 24 Breast Cancer Res Treat (2009) 114:23–30 independence using a genome-wide functional screen were done with FuGENE 6 (Roche Diagnostics GmbH, based on retrovirus-mediated insertion mutagenesis. Mannheim, Germany). Pools of transfectants were selected Insertion mutagenesis aims to introduce single genetic with G418 (Invitrogen) and subsequently assayed for modifications in the target cells in a random manner growth in the presence of 4-hydroxy-tamoxifen (OH-Tam, leading to an altered and selectable phenotype. Retrovi- Sigma–Aldrich Chemie BV, Zwijndrecht, The Nether- ruses are well suited for this purpose since they integrate in lands) as described previously [18–20]. The panel of a fairly random manner into the target cell genome as part tamoxifen-resistant cell lines was previously generated by of their life cycle. As a consequence, a particular target retrovirus infection and selection for OH-Tam resistance gene may become disrupted. More importantly, the pres- [17]. In brief, ZR-75-1 cells were infected with ampho- ence of strong promoter and enhancer elements in the tropic, defective murine retrovirus and plated in medium retroviral LTRs may increase the expression of genes in the containing 1 lM of OH-Tam. Within 5 weeks after start of immediate proximity [12]. Subsequently, retroviral selection, proliferating colonies were individually picked sequences may be used as a tag to isolate the target loci. and expanded to stable cell lines (further details in the The identification of a common Virus Integration Site Supplementary M&M, Text S1). (cVIS, i.e. a small region on the genome with multiple integration events in independent cell clones) strongly General molecular procedures indicates that the retrovirus inactivated or modulated expression of a target gene that underlies the selected To identify the retrovirus integration sites, we have per- phenotype. In recent years, large numbers of cVIS have formed inverse and Splinkerette PCR on high molecular been defined using high throughput technologies for vari- weight genomic DNA from these cell lines. The inverse PCR ous types of malignancies in mice [12–14]. procedure for circularized genomic DNA fragments has been The human ZR-75-1 breast cancer cell line is com- detailed previously [17, 21]. For the Splinkerette PCR [22], pletely dependent on estrogen for growth in tissue culture the restriction enzyme-specific adaptor was ligated to and a representative in vitro model for ER-positive breast digested genomic DNA. PCR was performed using primers cancer. Under anti-estrogen-supplemented culture condi- selected for the retrovirus, and for the Splinkerette adaptor. tions, ZR-75-1 cell proliferation is completely blocked. In Products were purified on agarose gel, sequenced and map- a proof of principle experiment, we have demonstrated ped on the human genome reference sequence assembly that the introduction of a single gene (EGFR) can trans- by BLAST or BLAT analyses (http://www.ncbi.nlm.nih. form this phenotype [15]. Moreover, random modulation gov/blast/ and http://genome.ucsc.edu/cgi-bin/hgGateway). of gene expression by DNA demethylation also caused Genes located in the immediate proximity of the integration estrogen-independent cell proliferation [16]. These results site were identified by manual inspection. Further details are provided the basis to initiate a genome-wide functional provided in the Text S1. screen using replication-defective murine retrovirus for For Northern blot analysis total RNA was isolated using the identification of BCAR genes contributing to estrogen- RNAzol B (Campro Scientific, Veenendaal, NL). Ten independent breast cancer cell proliferation in vitro [17]. microgram total RNA per lane was size fractioned on 1.2% In this manuscript, we describe the comprehensive anal- agarose-formaldehyde gel and transferred onto nitrocellu- ysis of the integration events of the defective retrovirus lose. Equivalent gel loading was judged from the Ethidium and the target genes responsible for estrogen indepen- Bromide-stained 28S and 18S rRNA bands and from dence of breast cancer cells. Furthermore, the relevance GAPD control hybridizations. Probes were derived from of these BCAR genes in human breast cancer is estab- cDNA clones or generated by gene-specific PCR, and lished. Our results document the power of such an sequence verified. Sequence analysis