Production of truncated MBD4 protein by frameshift mutation in DNA mismatch repair-deficient cells enhances 5-fluorouracil sensitivity that is independent of hMLH1 status

ABSTRACT

Methyl-CpG binding domain protein 4 (MBD4) is a DNA glycosylase that can remove 5-fluorodeoxyuracil from DNA as well as repair T:G or U:G mismatches. MBD4 is a target for frameshift mutation with DNA mismatch repair (MMR) deficiency, creating a truncated MBD4 protein (TruMBD4) that lacks its glycosylase domain. Here we show that TruMBD4 plays an important role for enhancing 5-fluorouracil (5FU) sensitivity in MMR-deficient colorectal cancer cells. We found biochemically that TruMBD4 binds to 5FU incorporated into DNA with higher affinity than MBD4. TruMBD4 reduced the 5FU affinity of the MMR recognition complexes that determined 5FU sensitivity by previous reports, suggesting other mechanisms might be operative to trigger cytotoxicity. To analyze overall 5FU sensitivity with TruMBD4, we established TruMBD4 overexpression in hMLH1-proficient or -deficient colorectal cancer cells followed by treatment with 5FU. 5FU-treated TruMBD4 cells demonstrated diminished growth characteristics compared to controls, independently of hMLH1 status. Flow cytometry revealed more 5FU-treated TruMBD4 cells in S phase than controls. We conclude that patients with MMR-deficient cancers, which show characteristic resistance to 5FU therapy, may be increased for 5FU sensitivity via secondary frameshift mutation of the base excision repair gene MBD4.

KEYWORDS:

• Colorectal cancer
• cytotoxicity
• DNA mismatch repair
• frameshift mutation
• MBD4
• truncated protein
• 5-fluorouracil

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Author contributions

MI and JMC conceived the study. SS and MI performed Western blotting and cell growth assays. MI and STR performed DNA pull down assays. SS, MI and HY constructed truncated MBD4 expression plasmid. SK, KS HM and JMC interpreted data. MI and JMC drafted and critically revised the manuscript. All authors read and approved the final manuscript.

Funding

Supported by the United States Public Health Service (DK067287, CA162147 and CA206010 to JMC) and Japan Society for the Promotion of Science (JSPS) (KAKENHI Grant Number 25860530 to MI).