Meijer Danielle, van Agthoven Ton, Bosma Peter T, Nooter Kees, Dorssers Lambert C J
Department of Pathology, Josephine Nefkens Institute, Erasmus MC-University Medical Center, Rotterdam, the Netherlands.
Mol Cancer Res. 2006 Jun;4(6):379-86. doi: 10.1158/1541-7786.MCR-05-0156.
Antiestrogens, such as tamoxifen, are widely used for endocrine treatment of estrogen receptor-positive breast cancer. However, as breast cancer progresses, development of tamoxifen resistance is inevitable. The mechanisms underlying this resistance are not well understood. To identify genes involved in tamoxifen resistance, we have developed a rapid screening method. To alter the tamoxifen-sensitive phenotype of human ZR-75-1 breast cancer cells into a tamoxifen-resistant phenotype, the cells were infected with retroviral cDNA libraries derived from human placenta, human brain, and mouse embryo. Subsequently, the cells were selected for proliferation in the presence of 4-hydroxy-tamoxifen (OH-TAM) and integrated cDNAs were identified by sequence similarity searches. From 155 OH-TAM-resistant cell colonies, a total of 25 candidate genes were isolated. Seven of these genes were identified in multiple cell colonies and thus cause antiestrogen resistance. The epidermal growth factor receptor, platelet-derived growth factor receptor-alpha, platelet-derived growth factor receptor-beta, colony-stimulating factor 1 receptor, neuregulin1, and fibroblast growth factor 17 that we have identified have been described as key regulators in the mitogen-activated protein kinase pathway. Therefore, this pathway could be a valuable target in the treatment of patients with breast cancer resistant to endocrine treatment. In addition, the putative gene LOC400500, predicted by in silico analysis, was identified. We showed that ectopic expression of this gene, designated as breast cancer antiestrogen resistance 4 (BCAR4), caused OH-TAM resistance and anchorage-independent cell growth in ZR-75-1 cells and that the intact open reading frame was required for its function. We conclude that retroviral transfer of cDNA libraries into human breast cancer cells is an efficient method for identifying genes involved in tamoxifen resistance.
抗雌激素药物，如他莫昔芬，被广泛用于雌激素受体阳性乳腺癌的内分泌治疗。然而，随着乳腺癌的进展，他莫昔芬耐药的出现是不可避免的。这种耐药的潜在机制尚未完全明确。为了鉴定与他莫昔芬耐药相关的基因，我们开发了一种快速筛选方法。为了将人ZR-75-1乳腺癌细胞的他莫昔芬敏感表型转变为耐药表型，将这些细胞用源自人胎盘、人脑和小鼠胚胎的逆转录病毒cDNA文库进行感染。随后，在4-羟基他莫昔芬（OH-TAM）存在的情况下选择细胞进行增殖，并通过序列相似性搜索鉴定整合的cDNA。从155个OH-TAM耐药细胞克隆中，共分离出25个候选基因。其中7个基因在多个细胞克隆中被鉴定出来，因此导致抗雌激素耐药。我们鉴定出的表皮生长因子受体、血小板衍生生长因子受体-α、血小板衍生生长因子受体-β、集落刺激因子1受体、神经调节蛋白1和成纤维细胞生长因子17已被描述为丝裂原活化蛋白激酶途径中的关键调节因子。因此，该途径可能是治疗内分泌治疗耐药乳腺癌患者的一个有价值的靶点。此外，通过电子分析预测的假定基因LOC400500也被鉴定出来。我们发现，该基因（命名为乳腺癌抗雌激素耐药4，BCAR4）的异位表达导致ZR-75-1细胞产生OH-TAM耐药和非锚定依赖性细胞生长，并且其完整的开放阅读框是其功能所必需的。我们得出结论，将cDNA文库逆转录病毒转移到人乳腺癌细胞中是鉴定与他莫昔芬耐药相关基因的一种有效方法。
