Reduced expression of MUTYH with suppressive activity against mutations caused by 8‐hydroxyguanine is a novel predictor of a poor prognosis in human gastric cancer

Abstract

The MUTYH gene encodes a DNA glycosylase that can initiate the excision repair of adenine mispaired with 8-hydroxyguanine (8OHG) and is responsible for a susceptibility to multiple colorectal adenomas and carcinomas. To determine whether the MUTYH gene is involved in gastric carcinogenesis, we first examined the expression level of MUTYH in gastric cancer. The reduced expression of MUTYH mRNA transcript was detected in both gastric cancer cell lines and primary gastric cancers using qRT-PCR analysis. Immunohistochemical analysis also showed a significant reduction in MUTYH protein expression in gastric cancer, compared with non-cancerous gastric epithelium (immunohistochemical score, 175.5 ± 43.0 versus 281.5 ± 24.8; p < 0.0001). Among the gastric cancers, the MUTYH expression level was significantly associated with the histopathology (p < 0.0001) and the pT stage (p < 0.001). The outcome of patients with gastric cancer exhibiting low MUTYH expression was significantly worse than the outcome of patients with gastric cancer exhibiting high MUTYH expression (p = 0.0007, log-rank test) and a multivariate analysis revealed that reduced MUTYH expression was an independent predictor of a poor survival outcome among the gastric cancer patients (hazard ratio, 1.865; 95% confidence interval, 1.028-3.529; p = 0.0401). We next compared the functional effects of MUTYH on gastric cancer cells, based on their MUTYH expression levels. MUTYH-over-expressing stable clones of the gastric cancer cell line AGS showed: (a) higher DNA cleavage activity towards adenine:8OHG mispair-containing substrates; (b) higher suppressive activity against mutations caused by 8OHG in a supF forward mutation assay; and (c) higher suppressive activity for cellular proliferation than empty vector-transfected AGS clones. These results suggested that MUTYH is a suppressor of mutations caused by 8OHG in gastric cells and that its reduced expression is associated with a poor prognosis in gastric cancer.

Mammalian MutY homolog (MYH or MUTYH) protects cells from oxidative DNA damage

“…Germline mutations in the hMYH gene is associated with human colorectal adenomatous polyposis in MAP patients [17-21] and down-regulation of MYH expression has been shown in human gastric cancer [49]. Cells harboring certain mutations in the hMYH gene are prone to G:C to T:A mutations in APC , K-ras , and other genes [17,22,23].…”

NEIL1 p.Gln282Stop variant is predominantly localized in the cytoplasm and exhibits reduced activity in suppressing mutations

“…Western blot analysis was performed as described previously, using a rabbit anti-GFP polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), a rabbit anti-FLAG polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), a rabbit anti-NEIL1 polyclonal antibody (Oncogene Research Products, Cambridge, MA, USA), a mouse anti-lamin A/C monoclonal antibody (3A6-4C11; Active Motif, Carlsbad, CA, USA), or a mouse anti-β-tubulin monoclonal antibody (2-28-33; SigmaAldrich) (Shinmura et al, 2011).…”

Impaired 8-Hydroxyguanine Repair Activity of MUTYH Variant p.Arg109Trp Found in a Japanese Patient with Early-Onset Colorectal Cancer

“…A Western blot analysis using a mouse anti-MUTYH monoclonal antibody (4D10; Abnova, Taipei, Taiwan) or an anti- β -tubulin monoclonal antibody (clone 2-28-33; Sigma-Aldrich, St. Louis, MO) was performed as described previously [22, 23]. …”

“…A supF forward mutation assay for H1299-derived cells was performed using the 8OHG-containing pMY189 plasmid and the KS40/pKY241 indicator E. coli strain, as described previously [22, 23]. The mutation frequencies were calculated as the number of E. coli supF mutants per total number of E. coli transformants.…”