Aberrant DNA methylation is associated with many types of human cancers. To identify genes silenced in human colorectal cancers, we performed a microarray analysis for genes whose expression was induced by treatment of HCT116 human colon cancer cells with a demethylating agent, 5-aza-2 0 -deoxycitidine (5-aza-dC). Seven known genes were identified as being upregulated (8-fold) and expressed at more than twice as high as the average level. Among these was the UCHL1 gene (also known as PGP9.5), which is involved in regulation of cellular ubiquitin levels. A dense CpG island in its promoter region was completely methylated in HCT116 cells, and no mRNA was detected. 5-Aza-dC treatment of HCT116 cells induced dose-dependent demethylation of the CpG island, and restored UCHL1 mRNA and protein expression. UCHL1 silencing was observed in 11 of 12 human colorectal cancer cell lines, and its methylation was detected in 8 of 17 primary colorectal cancers. Further, UCHL1 silencing was observed in 6 of 13 ovarian cancer cell lines, and its methylation was detected in 1 of 17 primary ovarian cancers. These results showed that UCHL1 is inactivated in human colorectal and ovarian cancers by its promoter methylation, and suggest that disturbance of cellular ubiquitin levels is present.

Alterations of epigenetic modifications are promising targets for cancer therapy, and several epigenetic drugs are now being clinically utilized. At the same time, individual epigenetic modifications have physiological functions in normal cells, and cancer cell specificity is considered difficult to achieve using a drug against a single epigenetic modification. To overcome this limitation, a combination of epigenetic modifications specifically or preferentially present in cancer cells is a candidate target. In this study, we aimed to demonstrate (i) the presence of a cancer cell-specific combination of epigenetic modifications by focusing on DNA methylation and trimethylation of histone H3 lysine 27 (H3K27me3) and (ii) the therapeutic efficacy of a combination of DNA demethylation and EZH2 inhibition. Analyses of DNA methylation and H3K27me3 in human colon, breast and prostate cancer cell lines revealed that 24.7±4.1% of DNA methylated genes had both DNA methylation and H3K27me3 (dual modification) in cancer cells, while it was 11.8±7.1% in normal cells. Combined treatment with a DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC) and an EZH2 inhibitor, GSK126, induced marked re-expression of genes with the dual modification, including known tumor-suppressor genes such as IGFBP7 and SFRP1, and showed an additive inhibitory effect on growth of cancer cells in vitro. Finally, an in vivo combined treatment with 5-aza-dC and GSK126 inhibited growth of xenograft tumors more efficiently than a single treatment with 5-aza-dC. These results showed that the dual modification exists specifically in cancer cells and is a promising target for cancer cell-specific epigenetic therapy.

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Tumor suppressors with extracellular function are likely to have advantages as targets for cancer therapy, but few are known. Here, we focused on angiopoietin-like 4 (ANGPTL4), which is a secreted glycoprotein involved in lipoprotein metabolism and angiogenesis, is methylation-silenced in human cancers, but has unclear roles in cancer development and progression. We found a deletion mutation in its coiled-coil domain at its N-terminal in human gastric cancers, in addition to hypermethylation of the ANGPTL4 promoter CpG islands. Forced expression of wild-type ANGPTL4, but not ANGPTL4 with the deletion, at physiological levels markedly suppressed in vivo tumorigenicity and tumor angiogenesis, indicating that the latter caused the former. Tumor-derived ANGPTL4 suppressed in vitro vascular tube formation and proliferation of human umbilical vascular endothelial cells, partly due to suppression of ERK signaling. These showed that ANGPTL4 is a genetically and epigenetically inactivated secreted tumor suppressor that inhibits tumor angiogenesis.

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Although conjugation of overexpressed GABARP to phospholipid has been reported during starvation-induced autophagy, it is unclear whether endogenous GABARAP-phospholipid conjugation is also activated under starvation conditions. We observed little accumulation of GABARAP-phospholipid conjugate (GABARAP-PL) in mouse liver and kidney under starvation conditions, whereas endogenous LC3-phospholipid conjugate (LC3-II) accumulated. A small amount of endogenous GABARAP-PL was observed in the heart, independent of starvation. In rapamycin-treated HEK293 cells, there was little accumulation of endogenous GABARAP-PL, even in the presence of lysosomal protease-inhibitors, whereas there was significant accumulation of endogenous LC3-II, together with inactivation of the mTor kinase-signaling pathway. In HeLa and C2C12 cells, GABARAP-PL accumulation in the presence of lysosomal protease inhibitors was independent of starvation-induced autophagy, whereas LC3-II accumulation was significant during starvation-induced autophagy. Interestingly, we observed activation of lysosomal turnover of GABARAP-PL during the differentiation of C2C12 cells to myotubes, along with increased lysosomal turnover of LC3-II. Under these conditions, S6 ribosomal protein was still phosphorylated, suggesting that the mTor kinase-signaling pathway is active during the differentiation of C2C12 cells to myotubes, in contrast to starvation-induced autophagy. These results indicated that lysosomal turnover of GABARAP-PL was activated during the differentiation of C2C12 cells to myotubes without inactivation of the mTor kinase-signaling pathway, whereas little lysosomal turnover of GABARAP-PL was activated during starvation-induced autophagy.

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