Silencing of the metastasis suppressor RECK by RAS oncogene is mediated by DNA methyltransferase 3b-induced promoter methylation.
RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase activity to suppress tumor invasion and metastasis. Our previous study indicated that oncogenic RAS inhibited RECK expression via a histone deacetylation mechanism. In this study, we address whether DNA methyltransferases (DNMT) participate in the inhibition of RECK by RAS. Induction of Ha-RAS(Val12) oncogene increased DNMT3b, but not DNMT1 and DNMT3a, expression in 2-12 cells. In addition, induction of DNMT3b by RAS was through the extracellular signal-regulated kinase signaling pathway. Oncogenic RAS increased the binding of DNMT3b to the promoter of RECK gene and this binding induced promoter methylation, which could be reversed by 5'-azacytidine and DNMT3b small interfering RNA (siRNA). The MEK inhibitor U0126 also reversed RAS-induced DNMT3b binding and RECK promoter methylation. Treatment of 5'-azacytidine and DNMT3b siRNA restored RECK expression in 2-12 cells and potently suppressed RAS-stimulated cell invasion. In addition, the inhibitory effect of 5'-azacytidine on RAS-induced cell invasion was attenuated after knockdown of RECK by siRNA. Interestingly, human lung cancer cells harboring constitutively activated RAS exhibited lower RECK expression and higher promoter methylation of RECK gene. 5'-Azacytidine and DNMT3b siRNA restored RECK expression in these cells and effectively suppressed invasiveness. Collectively, our results suggest that RAS oncogene induces RECK gene silencing through DNMT3b-mediated promoter methylation, and DNMT inhibitors may be useful for the treatment of RAS-induced metastasis.
Chang
Hui-Chiu
HC
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Cho
Chun-Yu
CY
Hung
Wen-Chun
WC
D016475:3T3 Cells; D000818:Animals; D002843:Chromatin / Q000502:physiology; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D058851:GPI-Linked Proteins; D020868:Gene Silencing; D011905:Genes, ras; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D014407:Tumor Cells, Cultured; D000097584:DNA Methyltransferase 3B
D002843:Chromatin; D004248:DNA (Cytosine-5-)-Methyltransferases; D004273:DNA, Neoplasm; D058851:GPI-Linked Proteins; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D000097584:DNA Methyltransferase 3B; C115460:RECK protein, human