Could a variant structural form of thymidylate synthase gene of metastatic colorectal cancer patients be related with the poor response to 5 Fu treatment?

Abstract

Background: Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of 5-fluorouracil (5-Fu). TS levels vary considerably among tumors and the response to 5-FU is influenced by the intratumoral activity of the enzyme, with high levels generally being associated with a poor response. Response to 5-FU also depends on TS structure and some researchers showed that variant structural forms of TS in tumour cell lines confer resistance to fluoropyrimidines so the aim our study is to analyze the whole coding regions of the TS gene to evaluate a possible mechanism of 5 Fu resistance. Materials and Methods: We performed the TS-DNA gene sequence in 68 colorectal cancer (CRC) samples from patients of different Dukes’ stages (A, B, C) and 13 metastatic samples (D). The sequence of the exons was performed amplified every exon by PCR, the amplification products of abour 180-310 bp were purified by kit Agencourt®CleanSEQ® della Beckman Coulter and sequenced by theABI3130 Genetic Analyzer della Applied Biosystems. Results. The sequencing of the 7 exons of the gene did not show any mutation in the coding region of patients at A, B, C dukes stage, while in metastatic ones was revealed a triple insertion (CCG) in the splicing region adjacent to 6 exon that could influence the splicing of the gene and as consequence the length of mRNA and the protein structure. Conclusions: 5-FU improves survival in a subgroup of CRC patients but predictive markers are required to identify patients that benefit from such a treatment, we found a mutation in the splicing region of the TS gene of metastatic CRCs that could explain their frequent 5-Fu drug resistance and a worse prognosis. A structural analysis of mRNA and protein will be necessary to valuate the real meaning of this insertion.