Functional Screen for Responsible for Tamoxifen Resistance in Human Breast Cancer Cells

Danielle Meijer,1 Ton van Agthoven,1 Peter T. Bosma,2 Kees Nooter,2 and Lambert C.J. Dorssers1

Departments of 1Pathology and 2Medical Oncology, Josephine Nefkens Institute, Erasmus MC-University Medical Center, Rotterdam, the Netherlands

Abstract intact open readingframe was required for its function. Antiestrogens, such as tamoxifen, are widely used for We conclude that retroviral transfer of cDNA libraries endocrine treatment of receptor–positive into human breast cancer cells is an efficient method for breast cancer. However, as breast cancer progresses, identifyinggenesinvolved in tamoxifen resistance. development of tamoxifen resistance is inevitable. The (Mol Cancer Res 2006;4(6):379–86) mechanisms underlyingthis resistance are not well understood. To identify genes involved in tamoxifen Introduction resistance, we have developed a rapid screening Tamoxifen is the most extensively used antiestrogen in the method. To alter the tamoxifen-sensitive phenotype treatment of breast cancer. Patients with estrogen receptor (ER)- of human ZR-75-1 breast cancer cells into a positive breast tumors may initially benefit from this treatment, tamoxifen-resistant phenotype, the cells were infected but almost all responding patients acquire resistance to the with retroviral cDNA libraries derived from human action of tamoxifen over time and the disease progresses. placenta, human brain, and mouse embryo. Several mechanisms for this phenomenon have been suggested, Subsequently, the cells were selected for proliferation in including alteration of the availability or metabolism of the presence of 4-hydroxy-tamoxifen (OH-TAM) and tamoxifen, alterations in the function of the ER and in the ER integrated cDNAs were identified by sequence similarity signaling cascade, and the altered expression of different genes searches. From 155 OH-TAM-resistant cell colonies, (reviewed by refs. 1, 2). However, in the majority of patients, a total of 25 candidate genes were isolated. Seven of the mechanisms causing tamoxifen-resistant proliferation re- these genes were identified in multiple cell colonies and main unexplained. Insight into these processes is essential for thus cause antiestrogen resistance. The epidermal the development of improved treatment strategies and may be growth factor receptor, platelet-derived growth factor obtained by the application of genome-wide functional screens. receptor-A, platelet-derived growth factor receptor-B, Random transfection of cDNA libraries was previously used colony-stimulatingfactor 1 receptor, neuregulin1, to identify the specific genes involved in progression to and fibroblast growth factor 17 that we have identified antiestrogen resistance of human breast cancer cells, but this have been described as key regulators in the had only limited success (3). On the other hand, our group has mitogen-activated kinase pathway. Therefore, successfully identified such genes using retroviral insertion this pathway could be a valuable target in the treatment mutagenesis in functional screens (4). Although insertion of patients with breast cancer resistant to endocrine mutagenesis has shown itself to be a very powerful tool in treatment. In addition, the putative LOC400500, the identification of genes involved in mouse tumorigenesis, in predicted by in silico analysis, was identified. our experiments the identification of the genes responsible for We showed that ectopic expression of this gene, antiestrogen-resistant proliferation has proved to be very labor designated as breast cancer antiestrogen resistance 4 intensive (5-7). (BCAR4), caused OH-TAM resistance and anchorage- To bypass this technical limitation, we have developed a independent cell growth in ZR-75-1 cells and that the rapid screening strategy for identifying gene