Abstract
1641
Background: Tamoxifen is the most extensively used anti-estrogen in the treatment of breast cancer. As breast cancer cells often develop tamoxifen resistance over time, the future development of new treatment strategies will require understanding of the mechanisms underlying this resistance. Objective: To identify genes and signaling cascades capable of inducing tamoxifen-resistant cell proliferation. Materials and Methods: In order to alter their estrogen-dependent phenotype into an estrogen-independent (tamoxifen-resistant) phenotype, ZR-75-1 breast cancer cells were transduced with retroviral cDNA expression libraries. These libraries were derived from either human placenta or mouse embryo. Subsequently, the cells were selected for proliferation in the presence of 4-hydroxy-tamoxifen (OH-TAM). Resistant cell clones were isolated and integrated cDNAs were amplified using PCR and identified by NCBI-database-similarity searches. Results: We isolated eighty OH-TAM-resistant cell clones from experiments with the placenta cDNA library, and an additional thirty cell clones from infection experiments with the embryo cDNA library. Our experiments revealed six genes capable of inducing OH-TAM resistance in multiple independent clones. Most of these genes have already been described as key regulators in signal transduction pathways, but we also identified a putative gene predicted by in-silico analysis of the human genome. We observed at least 26 independently derived cell clones containing this gene in three different screens with the human placenta library. Additional experiments were performed to validate its capacity to induce OH-TAM-resistant proliferation in human breast cancer cells. These experiments showed that, unlike control cells, ZR-75-1 cells expressing the putative gene are able to proliferate in the presence of the anti-estrogens OH-TAM and ICI182,780. Site-directed mutagenesis of this gene demonstrated that a frame-shift mutation interferes with stimulation of proliferation, indicating a causative role for the encoded protein. Conclusion: Retroviral transfer of cDNA libraries to human breast cancer cells is a very efficient method for identifying genes involved in tamoxifen resistance. The genes recovered in this screen may be part of specific pathways responsible for the tamoxifen-resistant proliferation of breast cancer cells. Ongoing experiments: In collaboration with the Department of Medical Oncology we are currently performing quantitative RT-PCR for the recovered genes to correlate their mRNA levels in tumors with clinical data of a large series of breast cancer patients. Acknowledgement: Supported by the Dutch Cancer Society.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]