Epigenetic silencing of CYP24 in the tumor microenvironment

Abstract

Calcitriol (1,25 dihydroxycholecalciferol) has significant anti-tumor activity in vitro and in vivo in a number of tumor model systems. We developed a system for isolation of fresh endothelial cells from tumors and Matrigel environments which demonstrate that CYP24, the catabolic enzyme involved in vitamin D signaling, is epigenetically silenced selectively in tumor-derived endothelial cells (TDEC). TDEC maintain phenotypic characteristics which are distinct from endothelial cells isolated from normal tissues and from Matrigel plugs (MDEC). In TDEC, calcitriol induces G(0)/G(1) arrest, modulates p27 and p21, and induces apoptotic cell death and decreases P-Erk and P-Akt. In contrast, endothelial cells isolated from normal tissues and MDEC are unresponsive to calcitriol-mediated anti-proliferative effects despite intact signaling through the vitamin D receptor (VDR). In TDEC, which are sensitive to calcitriol, the CYP24 promoter is hypermethylated in two CpG island regions located at the 5'end; this hypermethylation may contribute to gene silencing of CYP24. The extent of methylation in these two regions is significantly less in MDEC. Lastly, treatment of TDEC with a DNA methyltransferase inhibitor restores calcitriol-mediated induction of CYP24 and resistance to calcitriol. These data suggest that epigenetic silencing of CYP24 modulates cellular responses to calcitriol.

Figure 1

A and B. Calcitriol effect on the vasculature in vivo. (A) The effects of calcitriol (daily×3 0.625μg/mouse) on mean vessel density (MVD) in both SCC tumors and Matrigel plugs where 15 fields and 3 animals in each group were counted. (B) Apoptotic endothelial cells (dark staining) in SCC tumors and Matrigel plugs as determined by double staining of CD31 (endothelial cells) and caspase-3 (apoptosis) on frozen sections from 5 animals/group treated with calcitriol as described above.

Figure 2

A, B and C. CYP24 enzymes in TDEC and MDEC. (A) microarray screen of CYP enzymes in TDEC and MDEC, (B) schematic diagram showing the position of the two CpG islands at the promoter region of CYP24, (C) methylation analysis of CpG sites located at the CYP24 promoter using bisulfite sequencing. Each circle represents one CpG site.