Abstract 991: Integrated analysis of gene expression, DNA copy number, and CpG island methylation of 5-fluorouracil-resistant human gastric cancer cell lines.

Abstract

Abstract Background: A major problem in chemotherapy is treatment failure due to anticancer drug resistance. A better understanding of mechanisms underlying acquired resistance may facilitate the development of an optimal second-line therapy for recurrent cancer. This study was designed to elucidate mechanisms underlying acquisition of 5-fluoroucacil (5-FU) resistance by human gastric cancer cells. Method: 5-FU resistant cell lines were established by continuously exposing parent cell lines (MKN45, MKN-74, KATO-III, and NCI-N87) to escalating concentrations (1-5 μM) of 5-FU over 1 year. To elucidate mechanisms of resistance, gene expression, DNA copy number, and DNA methylation were measured by Whole Human Genome 44K oligo DNA microarray, 244K CGH microarray, and 244K CpG Island Microarray (Agilent Technologies, Santa Clara, CA), respectively. Data analysis was done with GeneSpring GX™ software, version 11.0 (Agilent Technologies) and Partek genomic suite software, version 6.5 (Partek Inc., St. Louis, MO). Results: Each of the cell lines acquired high resistance to 5-FU, although the degree of resistance varied. IC50 of the 5-FU-resistant cell lines was 3.8- to 11.6-fold higher than that of the parent cell lines. On gene expression analysis, 3.0%, 2.3%, 1.4%, and 1.4% of 41,093 probes were differentially expressed with more than 5-fold changes in MKN45/FU, KATOIII/FU, NCI-N87/FU, and MKN74/FU as compared with their parental cell lines, respectively. Changes occurring in at least 3 cell lines were expressed by only 0.2% of probes. Copy number analysis revealed that DNA copy gains occurred at 16.6%, 6.7%, 5.1%, and 5.6% of all genomic segments elucidated in MKN45/FU, KATOIII/FU, NCI-N87/FU and MKN74/FU, respectively. The frequencies of DNA copy losses were 35.6%, 14.0%, 3.2%, and 8.9%, respectively. Methylation analysis revealed that the number of methylated CpG sites in NCI-N87/FU and MKN74/FU increased by 1.5 times as compared with the parental cell lines. In contrast, the numbers of methylated CpG sites in MKN45/FU and KATOIII/FU rarely changed (around 40% each). Conclusions: Mechanisms leading to acquired resistance against 5-FU differed among the 4 gastric cancer cell lines. Changes in copy number most frequently occurred in MKN45/FU cells. In NCI-N87/FU and MKN74/FU, the numbers of methylated CpG sites increased. Such genome-wide integrated analysis will facilitate an understanding of mechanisms for 5-FU acquired resistance and help to identify therapeutic target genes. Citation Format: Takashi Kobunai, Ayako Nakamura, Mamoru Nukatsuka, Kazuhiko Hayashi, Teiji Takechi. Integrated analysis of gene expression, DNA copy number, and CpG island methylation of 5-fluorouracil-resistant human gastric cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 991. doi:10.1158/1538-7445.AM2013-991