Genome-wide screening of loci associated with drug resistance to 5-fluorouracil-based drugs

Abstract

Resistance to chemotherapeutic agents represents the chief cause of mortality in cancer patients with advanced disease. Chromosomal aberration and altered gene expression are the main genetic mechanisms of tumor chemoresistance. In this study, we have established an algorithm to calculate DNA copy number using the Affymetrix 10K array, and performed a genome-wide correlation analysis between DNA copy number and antitumor activity against 5-fluorouracil (5-FU)-based drugs (S-1, tegafur + uracil [UFT], 5'-DFUR and capecitabine) to screen for loci influencing drug resistance using 27 human cancer xenografts. A correlation analysis confirmed that the single nucleotide polymorphism (SNP) showing significant associations with drug sensitivity were concentrated in some cytogenetic regions (18p, 17p13.2, 17p12, 11q14.1, 11q11 and 11p11.12), and we identified some genes that have been indicated their relations to drug sensitivity. Among these regions, 18p11.32 at the location of the thymidylate synthase gene (TYMS) was strongly associated with resistance to 5-FU-based drugs. A change in copy number of the TYMS gene was reflected in the TYMS expression level, and showed a significant negative correlation with sensitivity against 5-FU-based drugs. These results suggest that amplification of the TYMS gene is associated with innate resistance, supporting the possibility that TYMS copy number might be a predictive marker of drug sensitivity to fluoropyrimidines. Further study is necessary to clarify the functional roles of other genes coded in significant cytogenetic regions. These promising data suggest that a comprehensive DNA copy number analysis might aid in the quest for optimal markers of drug response.

Figure 1

DNA copy number profiles for SC‐2, Lu99 and St‐40 human xenografts. The diagram shows the log₂ values of the ratios of the DNA copy numbers with respect to their genomic order from 1pter (left) to Xqter (right) for (a) SC‐2, (b) Lu99 and (c) St‐40, based on their physical positions. The log₂ ratio of 0 represents two copies. These copy numbers were calculated using virtual reference data (see Materials and methods), and 0 on the y‐axis represents two copies, which is the normal number of copies for autosomal genes. The aberrant loci indicated by the arrows are located in (a) MYC, (b) CDKN2A and (c) ERBB2. Chromosomal boundaries are displayed as vertical lines.

Figure 2

Validation of thymidylate synthase gene (TYMS) copy number. Correlations between TYMS copy number measured by real‐time polymerase chain reaction and inferred by (a) 10K array, (b) TYMS mRNA level and (c) thymidylate synthase protein level. Pearson correlation coefficients, r, and P‐values are shown. ^alog₂ ratio of TYMS copy number inferred by 10K array. ^bMeasured by quantitative real‐time PCR with reference to Line‐1 control. The log₂ ratio of 0 for copy number represents two copies.