Abstract
Resistance to 5-Fluoruracil (5-FU) has been linked to elevated expression of the main target, thymidylate synthase (TYMS), which catalyses the de novo pathway for production of deoxythymidine monophosphate. The potent oncogenic forkhead box transcription factor, FOXM1 is is regulated by E2F1 which also controls TYMS. This study reveals a significant role of FOXM1 in 5-FU resistance. Overexpression and knock-down studies of FOXM1 in colon cancer cells suggest the importance of FOXM1 in TYMS regulation. ChIP and global ChIP-seq data also confirms that FOXM1 can also potentially regulate other 5-FU targets, such as TYMS, thymidine kinase 1 (TK-1) and thymidine phosphorylase (TYMP). In human colorectal cancer tissue specimens, a strong correlation of FOXM1 and TYMS staining was observed. Elevated FOXM1 and TYMS expression was also observed in acquired 5-FU resistant colon cancer cells (HCT116 5-FU Res). A synergistic effect was observed following treatment of CRC cells with an inhibitor of FOXM1, thiostrepton, in combination with 5-FU. The combination treatment decreased colony formation and migration, and induced cell cycle arrest, DNA damage, and apoptosis in CRC cell lines. In summary, this research demonstrated that FOXM1 plays a pivotal role in 5-FU resistance at least partially through the regulation of TYMS.
Highlights
Colorectal cancer (CRC) is a leading cause of cancer mortality, andcurrent strategies for treating this condition need to be improved[1,2]
We further evaluated thymidylate synthase enzyme (TYMS) expression in the same cohort and observed strong TYMS positive staining in the cytoplasm of CRC cells
There was a significant correlation between FOXM1 and TYMS (Spearman r = 0.314, p = 0.008)
Summary
TYMS expression and its direct association with FOXM1 in patients with colon cancer. To study the expression and correlation of FOXM1 and TYMS in colon cancer, immunohistochemistry was performed in a commercial colorectal tumour tissue microarray of 110 colon cancer samples (Fig. 1A). In p53 wt HCT116 cells, an initial induction of FOXM1, TYMS, and E2F1 expression was observed followed by a decrease at later time-points; in p53 mutant DLD1 and HT29 colorectal cancer cells these proteins remain elevated, which is consistent with previous studies demonstrating that p53 is required for the downregulation of FOXM1 acting through p21Cip[125] and DNA-damage strongly up-regulated the level of FOXM1 in the absence of functional p5319,25. (ABCC10) expression in colorectal cancer cells[32] This mechanism will no doubt contribute to the global role of FOXM1 in mediating 5-FU resistance; it is notable from our work that upon 5-FU treatment (1 μg/ mL) (Fig. 2E), all the TYMS proteins were converted into the inactive and slower migrating FdUMP-ligated forms in the sensitive HCT116 colon carcinoma cell lines, whereas in the resistant HCT116 5-FU Res cells a great proportion of the overexpressed TYMS proteins remained active and uncomplexed[33]. FOXM1 could be a novel predictive biomarker in 5-FU therapy, and new treatment strategies that target FOXM1 should be developed to improve clinical response in CRC
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