Identification of epigenetically silenced genes in tumor endothelial cells.
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.
Hellebrekers
Debby M E I
DM
Department of Pathology, Research Institute for Growth and Development, Maastricht University and University Hospital, Maastricht, the Netherlands.
Melotte
Veerle
V
Viré
Emmanuelle
E
Langenkamp
Elise
E
Molema
Grietje
G
Fuks
François
F
Herman
James G
JG
Van Criekinge
Wim
W
Griffioen
Arjan W
AW
van Engeland
Manon
M
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D018938:Caco-2 Cells; D051152:Clusterin / Q000235:genetics; D003110:Colonic Neoplasms / Q000098:blood supply* / Q000235:genetics; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D042783:Endothelial Cells / Q000187:drug effects / Q000473:pathology / Q000502:physiology*; D044127:Epigenesis, Genetic; D000071838:Fibrillin-1; D000071837:Fibrillins; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008840:Microfilament Proteins / Q000235:genetics; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000473:pathology; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013879:Thioredoxins / Q000235:genetics; D054392:Adipokines
D001374:Azacitidine; D051152:Clusterin; D015254:DNA Modification Methylases; D000077209:Decitabine; D000071838:Fibrillin-1; D000071837:Fibrillins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D008840:Microfilament Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; D013879:Thioredoxins; C495242:CLU protein, human; C000605722:FBN1 protein, human; D054392:Adipokines; C012589:trichostatin A; C117183:QSOX1 protein, human