Abstract 5018: Transcriptional and functional analysis of the CHD5 gene in Colorectal Cancer

Abstract

Abstract Background: CHD5 is a family member of chromatin remodeling factors that functions as a tumor suppressor in neuroblastoma and ovarian cancer. The transcriptional activity of CHD5 is silenced by hypermethylation. It has been found that CHD5 is a biomarker in colon carcinogenesis. In this study, we have investigated the transcriptional regulation and function of the CHD5 in CRC. Methods: The transcriptional activity of CHD5 was tested by reporter luciferase assay. The 1kb large fragment upstream of CHD5 transcription start site (TSS) showed luciferase activity; while the 400 bp subfragment upstream of TSS was inactive in both RKO (CHD5 endogenous non-expressing) and HCT116 (CHD5 endogenous expressing) cell lines. This suggests that the transcriptionally active sites of CHD5 are located within −400 to −1000. There are two consensus TCF binding sites in the 1 kb promoter region. In order to investigate whether Wnt pathway affects the CHD5 transcriptional activity, we treated the RKO and HCT116 cell lines with 6-bromoindirubin-3′oxime (BIO), the GSK-3a inhibitor, which activates the β-Catenin. We tested the Wnt pathway activity in RKO and HCT116 cell lines by using the TCF/LEF responsive luciferase construct. Methylation profiles of CHD5 in 51 CRC and 6 adenomas from African Americans analyzed by MSP. Microdeletion analysis of the CHD5 region (1p36) was performed by aCGH. Results: The activity of TCF/LEF responsive element increased by 17.5 folds in RKO cell line and by 7.3 in HCT116 cell line after BIO treatment. Interestingly, the luciferase activity of 1kb CHD5 promoter increased after treating with BIO, and no changes were observed in activity of the 400 bp fragment. This suggests that the TCF/LEF and β-Catenin might act as coactivators for CHD5 transcriptional regulation. We also analyzed the proliferation of the RKO cell line after stably transfecting with CHD5 expressing plasmid (pcDNA). The results showed that the exogenous expression of CHD5 reduces the proliferation of the RKO cell line by two folds compared to the wild type and the mock controls. CHD5 was hypermethylated in 78% of the tested patients samples and deleted in 36%. Conclusion: The function of CHD5 is not well understood in CRC. Here, we suggest that the wnt/β-catenin pathway may be involved in the transcriptional regulation of CHD5. In addition in vivo CHD5 microdeletion and methylation excrement suggest that CHD5 act as tumor suppressor in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5018.