Vitamin D in Prostate Cancer: Tumor Vasculature Effects

描述（由申请人提供）：骨化三醇（维生素D或1,25二羟基胆骨化醇）是骨骼和矿物质代谢的中心因子，在体外和体内具有显著的抗肿瘤活性。诱导主要负责骨化三醇分解代谢的酶CYP24可能是临床前和临床抗增殖活性的一个因素。此外，肿瘤内皮细胞对骨化三醇敏感，并通过表观遗传改变独特地调节CYP24的表达。表观遗传事件在不改变DNA基因序列的情况下影响基因表达，导致肿瘤抑制基因的转录基因沉默和失活。虽然许多研究记录了肿瘤细胞的表观遗传变化，但只有有限的数据支持表观遗传变化在肿瘤微环境中发现的“正常”细胞中的作用。骨化三醇还可以抑制内皮细胞的增殖，并在许多肿瘤模型系统中抑制血管生成。我们已经建立了一种分离新鲜的，肿瘤来源的内皮细胞（TDEC）的方法，这些细胞保持着不同于从正常组织和基质塞（MDEC）中分离的内皮细胞的表型特征。在TDEC中，骨化三醇诱导G0/G1阻滞，调节p27和p21，诱导凋亡细胞死亡，降低P-Erk和P-Akt。相比之下，从正常组织和MDEC分离的内皮细胞对骨化三醇介导的抗增殖作用没有反应，尽管通过维生素D受体（VDR）完整的信号传导。差异可能是由于CYP24在MDEC中过度表达，CYP24的mRNA、蛋白和酶活性显著增加。在对骨化三醇敏感的TDEC中，CYP24启动子在位于5'端的两个CpG岛区高甲基化，这可能导致了TDEC中CYP24的基因沉默。在MDEC中，这两个区域的甲基化程度明显较低。DNA甲基转移酶抑制剂5-aza-2'-脱氧胞苷治疗TDEC，恢复骨化三醇介导的CYP24诱导和对骨化三醇的抗性。最后，当MDEC与肿瘤细胞的条件培养基孵育14-21天时，这些细胞恢复对骨化三醇抗增殖作用的敏感性，CYP24的诱导被沉默，CYP24启动子的1区和2区被超甲基化。这些细胞的特征与肿瘤或TDEC中发现的内皮细胞相似。肿瘤微环境中骨化三醇诱导内皮细胞CYP24基因表达的表观遗传沉默的这些独特差异直接影响骨化三醇介导的信号通路并最终影响治疗应用。因此，我们建议：1)阐明导致CYP24表观遗传沉默的表观遗传信号转导机制；2)确定CYP24的表观遗传沉默是否发生在人前列腺癌分离的内皮细胞和肿瘤细胞群中；3)通过研究内皮细胞肿瘤微环境中表观遗传缺陷的范围，确定TDEC与MDEC中DNA甲基化的其他潜在靶点。我们开发了一个从肿瘤中分离内皮细胞的模型系统，并证明CYP24，一种参与维生素D（1,25二羟基胆骨化醇或骨化三醇）信号传导的分解代谢酶，在肿瘤来源的内皮细胞中选择性地变得表观遗传沉默。肿瘤微环境中骨化三醇诱导内皮细胞CYP24基因表达的表观遗传沉默的这些独特差异直接影响骨化三醇介导的信号通路并最终影响治疗应用。

DESCRIPTION (provided by applicant): Calcitriol (vitamin D or 1,25 dihydroxycholecalciferol), a central factor in bone and mineral metabolism, has significant antitumor activity in vitro and in vivo. Induction of CYP24, the enzyme primarily responsible for calcitriol catabolism, may be a factor in the anti-proliferative activity pre-clinically and clinically. In addition, the endothelial cells in tumors are sensitive to calcitriol and uniquely modulate CYP24 expression through epigenetic changes. Epigenetic events affect gene expression without alteration in DNA gene sequence and lead to transcriptional gene silencing and inactivation of tumor suppressor genes in human cancer. While many studies document epigenetic changes in tumor cells, only limited data support a role for epigenetic changes in the "normal" cells found in the tumor microenvironment. Calcitriol also inhibits proliferation of endothelial cells and can inhibit angiogenesis in a number of tumor model systems. We have established a method for the isolation of fresh, tumor-derived endothelial cells (TDEC) that maintain phenotypic characteristics which are distinct from endothelial cells isolated from normal tissues and from Matrigel plugs (MDEC). In TDEC, calcitriol induces G0/G1 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). Differences may be due to the over-expression of CYP24 in MDEC where mRNA, protein and enzymatic activity for CYP24 are markedly increased. In TDEC, which is sensitive to calcitriol, the CYP24 promoter is hypermethylated in two CpG island regions located at the 5' end, which may contribute to gene silencing of CYP24 in TDEC. The extent of methylation in these two regions is significantly less in MDEC. Treatment of TDEC with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restores calcitriol-mediated induction of CYP24 and resistance to calcitriol. Lastly, when MDEC are incubated with conditioned media from tumor cells for 14-21 days, these cells regain sensitivity to the antiproliferative effects of calcitriol, CYP24 induction is silenced and regions 1 and 2 of the CYP24 promoter are hypermethylated. The characteristics of these cells are similar to the endothelial cells found in tumors or TDEC. These unique differences in the epigenetic silencing of calcitriol-induced CYP24 gene expression in endothelial cells from the tumor microenvironment directly impact on calcitriol-mediated signaling pathways and ultimately on therapeutic application. Therefore, we propose 1) to elucidate the epigenetic signal transduction mechanism(s) that lead to the epigenetic silencing of CYP24; 2) to determine whether epigenetic silencing of CYP24 occurs in human endothelial cell and tumor cell populations isolated from human prostate cancer; and 3) To identify other potential targets of DNA methylation in TDEC vs. MDEC by investigating the scope of the epigenetic defect from the tumor microenvironment on the endothelial cells. We developed a model system for isolation of endothelial cells freshly from tumors and demonstrate that CYP24, the catabolic enzyme involved in vitamin D (1,25 dihydroxycholecalciferol or calcitriol) signaling, becomes epigenetically silenced selectively in tumor-derived endothelial cells. These unique differences in the epigenetic silencing of calcitriol-induced CYP24 gene expression in endothelial cells from the tumor microenvironment directly impact on calcitriol-mediated signaling pathways and ultimately on therapeutic application.