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

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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.