Isolation of mammary cancer stem cell and analysis of its molecular biology for targeted chemotherapy in breast cancer

本研究的目的是从乳腺肿瘤组织中分离出一种乳腺癌干细胞（肿瘤起始细胞）和一种非肿瘤起始细胞，并对其分子特性进行表征，为乳腺癌干细胞的靶向化疗奠定基础。由于5-氟尿嘧啶（5-FU）通常用于乳腺癌患者，我们将5-FU的靶酶胸苷酸合成酶（TS）作为5-FU化疗的预测标志物。首先，我们用PCR-RFLP方法分析了37对正常乳腺组织、原发肿瘤和转移淋巴结中3 '-非翻译区（UTR）1494位核苷酸的TS多态性。其次，采用ELISA法检测了37例正常乳腺组织、乳腺癌原发灶和转移淋巴结组织中TS蛋白的表达。在2例正常乳腺组织中发现TS基因第1494位核苷酸的6个碱基缺失（del/del型）。在13例正常组织中发现1个等位基因缺失（del/ins型）。22例正常组织中TS基因3 '-UTR无缺失（ins/ins型）。在13例癌组织和15例转移性淋巴结中，del/del型多态性增加。我们研究了TS基因多态性类型与TS蛋白表达水平在同一患者原发肿瘤和转移淋巴结中的关系。TS基因3'非翻译区ins/ins型多态性的原发性肿瘤TS蛋白表达显著高于TS基因3'非翻译区其他类型多态性的原发性肿瘤。这些结果提示TS基因3' UTR多态性对氟嘧啶敏感性可能有一定的影响。

The aim of our study is to isolate a breast cancer stem cell (tumor initiating cell) and a non-tumor initiating cell from breast tumor tissues, and to characterize molecular properties of those cells, in order to establish target chemotherapy on breast cancer stem cells. Since 5-fluorouracil (5-FU) is usually given to breast cancer patients, we focused on thymidylate synthase (TS) which is a target enzyme for 5-FU as a predictive marker of chemotherapy with 5-FU. First, we analyzed TS polymorphisms at the nucleotide 1494 in 3'-untranslated region (UTR) by PCR-RFLP, in 37 pairs of the normal breast tissue, primary tumor, and metastatic lymph node. Secondly, we evaluated the amount of TS protein by ELISA assay in 37cases of the normal breast tissue, primary tumor, and metastatic lymph node, respectively. Deletions of 6 base pairs at the nucleotide 1494 on both alleles of TS gene (del/del type) were found in normal breast tissues of 2 cases. Deletion of 6-bp on one allele (del/ins type) were observed in 13 normal tissues. Twenty-two normal tissues had no deletion in 3'-UTR of TS gene (ins/ins type). Polymorphism of del/del type was increased up to 13 cases of carcinoma tissues and up to 15 cases of a metastatic lymph node. We studied the relationship between polymorphic types of TS gene and the level of TS protein expression in a primary tumor and a metastatic lymph node from the same patient. A primary tumor with polymorphism of ins/ins type in 3' UTR of TS gene had a significantly higher expression of TS protein than a primary tumor with other types of polymorphism in 3' UTR of TS gene. These results suggest a potential role of polymorphism in 3' UTR of TS gene on fluoropyrimidine sensitivity.