Hellebrekers Debby M E I, Melotte Veerle, Viré Emmanuelle, Langenkamp Elise, Molema Grietje, Fuks François, Herman James G, Van Criekinge Wim, Griffioen Arjan W, van Engeland Manon
Department of Pathology, Research Institute for Growth and Development, Maastricht University and University Hospital, Maastricht, the Netherlands.
Cancer Res. 2007 May 1;67(9):4138-48. doi: 10.1158/0008-5472.CAN-06-3032.
Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated by DNMTs and HDAC are involved in regulation of endothelial cell gene expression during tumor angiogenesis. To understand the mechanisms behind the epigenetic regulation of tumor angiogenesis, we used microarray analysis to perform a comprehensive screen to identify genes down-regulated in tumor-conditioned versus quiescent endothelial cells, and reexpressed by 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA). Among the 81 genes identified, 77% harbored a promoter CpG island. Validation of mRNA levels of a subset of genes confirmed significant down-regulation in tumor-conditioned endothelial cells and reactivation by treatment with a combination of DAC and TSA, as well as by both compounds separately. Silencing of these genes in tumor-conditioned endothelial cells correlated with promoter histone H3 deacetylation and loss of H3 lysine 4 methylation, but did not involve DNA methylation of promoter CpG islands. For six genes, down-regulation in microdissected human tumor endothelium was confirmed. Functional validation by RNA interference revealed that clusterin, fibrillin 1, and quiescin Q6 are negative regulators of endothelial cell growth and angiogenesis. In summary, our data identify novel angiogenesis-suppressing genes that become silenced in tumor-conditioned endothelial cells in association with promoter histone modifications and reactivated by DNMT and HDAC inhibitors through reversal of these epigenetic modifications, providing a mechanism for epigenetic regulation of tumor angiogenesis.
肿瘤血管生成需要对内皮细胞中的基因表达进行复杂的调控。我们最近发现，DNA甲基转移酶（DNMT）和组蛋白脱乙酰酶（HDAC）抑制剂可直接抑制内皮细胞生长和肿瘤血管生成，这表明由DNMTs和HDAC介导的表观遗传修饰参与了肿瘤血管生成过程中内皮细胞基因表达的调控。为了了解肿瘤血管生成表观遗传调控背后的机制，我们使用微阵列分析进行了全面筛选，以鉴定在肿瘤条件下的内皮细胞与静止内皮细胞中下调且能被5-氮杂-2'-脱氧胞苷（DAC）和曲古抑菌素A（TSA）重新表达的基因。在鉴定出的81个基因中，77%含有启动子CpG岛。对一部分基因的mRNA水平进行验证，证实了在肿瘤条件下的内皮细胞中这些基因显著下调，并且通过DAC和TSA联合处理以及单独使用这两种化合物处理后可重新激活。在肿瘤条件下的内皮细胞中沉默这些基因与启动子组蛋白H3去乙酰化以及H3赖氨酸4甲基化的丧失相关，但不涉及启动子CpG岛的DNA甲基化。对于六个基因，在显微切割的人肿瘤内皮细胞中的下调得到了证实。通过RNA干扰进行的功能验证表明，聚集素、原纤蛋白1和静息素Q6是内皮细胞生长和血管生成的负调节因子。总之，我们的数据鉴定出了新的血管生成抑制基因，这些基因在肿瘤条件下的内皮细胞中因启动子组蛋白修饰而沉默，并通过DNMT和HDAC抑制剂逆转这些表观遗传修饰而重新激活，为肿瘤血管生成的表观遗传调控提供了一种机制。
