Abstract LB-220: Forkhead box transciption factor M1 (FOXM1) plays a critical role in colorectal cancer resistance by regulating thymidylate synthase (TS)

Abstract

Background: Tumor resistance to chemotherapy is a major challenge in cancer therapeutics today. 5-Fluorouracil (5-FU) is one of the most commonly used cancer therapeutic agents. The Forkhead box transcription factor M1 (FOXM1) has been shown to play vital roles in cell cycle progression, apoptosis, and cell survival. FOXM1 is over expressed in many types of cancers, including colorectal carcinoma and has been implicated in drug resistance in breast cancer. Thymidylate synthase (TS) enzyme is an important precursor in DNA synthesis. The mechanism of action of 5-FU has been associated with TS. High TS expression levels in tumors are generally related to 5-FU resistance. In this study we investigated the role of FOXM1 in 5-FU resistance and the therapeutic potential of the FOXM1 inhibitor thiostrepton (a thiazole antibiotic) in combination with 5-FU in colorectal cancer. We further analyzed the relation between TS and FOXM1 gene expressions in colorectal cancer.

Methods and Results: HCT116, DLD1, and HT29 cells were treated with 5-FU to detect the protein, mRNA expression of FOXM1 and TS. This resulted in a decrease in TS and FOXM1 expression in p53 wild type HCT116 cells but not in p53 mutant DLD1 or HT29. We also observed that FOXM1 expression was significantly correlated with TS expression by thiostrepton treatment. The IC50 of 5-FU was determined by SRB assay and was found to be 0.92ug/mL, 0.45ug/mL and 2.8ug/mL, while thiostrepton IC50 was found to be 0.60uM, 1.07uM, and 3.54uM, in HCT116, DLD1, and HT29 cells respectively. Combination of 5-FU and thiostrepton showed synergistic effect in all 3 colorectal cancer cells. Chromatin immunoprecipitation (ChIP) was performed in HCT116 and DLD1 cells to determine if FOXM1 binds to the TS promoter region (0 - 2000bp upstream of the TS transcription start site). Three negative primers were designed further to 2000bp upstream region. ChIP data revealed enrichment for FOXM1 at the TS promoter (0 - 1500 bp upstream) and no enrichment observed in the negative control. Inhibition of FOXM1 by thiostrepton led to a decrease in enrichment in the TS promoter region. These results confirmed the role of FOXM1 in TS regulation. Chip Sequencing (ChIP-seq) was then performed to study further FOXM1 targets and binding sites of FOXM1 to other well-known 5-FU targets such as thymidine phosphorylase (TP) and thymidine kinase 1 (TK1). The FOXM1 specific ChIP-seq libraries and corresponding input controls were sequenced on the Illumina Hi Seq 2000. ChIP-seq confirmed that FOXM1 significantly binds to both these factors. FOXM1 binding to cell cycle regulatory genes E2F1, E2F2 and E2F3 was also shown in both cell lines.

Conclusion: FOXM1 plays a critical role in 5-FU drug resistance by regulating 5-FU target genes such as TS, TK1. Inhibition of FOXM1 acts synergistically with 5-FU in colorectal cancer and warrants further investigation as a new treatment strategy.

Citation Format: Vidhya Varghese, Luca Magnani, Narumi Harada, Francesco A. Mauri, Eric W. Lam, Laura M. Kenny. Forkhead box transciption factor M1 (FOXM1) plays a critical role in colorectal cancer resistance by regulating thymidylate synthase (TS). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-220. doi:10.1158/1538-7445.AM2014-LB-220