Background/Aims: Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor lesion of colon cancer. Although the relevance of DNA hypermethylation in the SSA/P-cancer sequence is well documented, the role of DNA hypomethylation is unknown. We investigated the biological relevance of DNA hypomethylation in the SSA/P-cancer sequence by using 3-dimensional organoids of SSA/P. Methods: We first analyzed hypomethylated genes using datasets from our previous DNA methylation array analysis on 7 SSA/P and 2 cancer in SSA/P specimens. Expression levels of hypomethylated genes in SSA/P specimens were determined by RT-PCR and immunohistochemistry. We established 3-dimensional SSA/P organoids and performed knockdown experiments using a lentiviral shRNA vector. DNA hypomethylation at CpG sites of the gene was quantitated by MassARRAY analysis. Results: The mean number of hypomethylated genes in SSA/P and cancer in SSA/P was 41.6 ± 27.5 and 214 ± 19.8, respectively, showing a stepwise increment in hypomethylation during the SSA/P-cancer sequence. S100P, S100α2, PKP3, and MUC2 were most commonly hypomethylated in SSA/P specimens. The mRNA and protein expression levels of S100P, S100α2, and MUC2 were significantly elevated in SSA/P compared with normal colon tissues, as revealed by RT-PCR and immunohistochemistry, respectively. Among these, mRNA and protein levels were highest for S100P. Knockdown of the S100P gene using a lentiviral shRNA vector in 3-dimensional SSA/P organoids inhibited cell growth by >50% (p < 0.01). The mean diameter of SSA/P organoids with S100P gene knockdown was significantly smaller compared with control organoids. MassARRAY analysis of DNA hypomethylation in the S100P gene revealed significant hypomethylation at specific CpG sites in intron 1, exon 1, and the 5′-flanking promoter region. Conclusion: These results suggest that DNA hypomethylation, including S100P hypomethylation, is supposedly associated with the SSA/P-cancer sequence. S100P overexpression via DNA hypomethylation plays an important role in promoting cell growth in the SSA/P-cancer sequence
