Genomic Screening for Genes Silenced by DNA Methylation Revealed an Association between RASD1 Inactivation and Dexamethasone Resistance in Multiple Myeloma

Abstract

Purpose: Epigenetic changes such as DNA methylation play a key role in the development and progression of multiple myeloma. Our aim in the present study was to use genomic screening to identify genes targeted for epigenetic inactivation in multiple myeloma and assess their role in the development of resistance to dexamethasone. Experimental Design: Gene expression was examined using microarray screening, reverse transcription-PCR, and real-time quantitative PCR. DNA methylation was examined using bisulfite PCR, bisulfite sequencing, and bisulfite pyrosequencing in 14 multiple myeloma cell lines, 87 multiple myeloma specimens, and 12 control bone marrow samples.WST-8 assays were used to assess cell viability after treatment with 5-aza-2 ¶-deoxycytidine and/or dexamethasone. Results: Microarray analysis was done to screen for genes up-regulated by 5-aza-2 ¶-deoxycytidine. In RPMI8226 cells, 128 genes were up-regulated, whereas 83 genes were up-regulated in KMS12PE cells. Methylation of 22 genes with CpG islands in their 5 ¶ regions, including RASD1, was confirmed. Methylation of RASD1was associated with its inactivation, which correlated with resistance to dexamethasone. Treating multiple myeloma cells with 5-aza-2 ¶-deoxycytidine restored sensitivity to dexamethasone. Methylation of RASD1 was also detected in a subset of primary multiple myeloma specimens, and the levels of methylation were increased after repeated antitumor treatments. Gene signature analysis revealed various genes to be synergistically induced by treatment with a combination of 5-aza-2 ¶-deoxycytidine plus dexamethasone. Conclusion: Our findings indicate that epigenetic inactivation of genes, including RASD1, plays a key role in the development of dexamethasone resistance in multiple myeloma. Moreover, they show the utility of demethylation therapy in cases of advanced multiple myeloma.

DNA Methylation Analysis Determines the High Frequency of Genic Hypomethylation and Low Frequency of Hypermethylation Events in Plasma Cell Tumors

“…Several studies have reported hypermethylation in MM for specific genes, including SPARC, BNIP3, and TGFBR2 (5,20). Other genes with reported hypermethylation in MM are VHL, XAF1, EGLN1, EGLN3, CDKN2A, CCND2, TP53, DCC ESR1, CDH1, and CDKN2B, among others (6,19,24,25,27,(33)(34)(35)(36)(37). A number of reasons might explain the discordance in ours versus these other studies.…”

“…One of the most widely studied and understood epigenetic modifications involves methylation of CpG dinucleotides. Relatively little is still known about the degree to which CpG methylation contributes to myelomagenesis, and to date most studies have focused on the analyses of single genes (6,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Thus, the goal of this study is to perform a DNA methylation profiling analysis comparing CD138 + sorted MM and its precursor stages, MGUS and SMM, to NPC to identify important DNA methylation events associated with each tumor stage and tumor progression.…”

Ginkgolide B promotes proliferation and functional activities of bone marrow-derived endothelial progenitor cells: involvement of Akt/eNOS and MAPK/p38 signaling pathways

“…The effect of GB on EPCs proliferation was determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl) -2H-tetrazolium, monosodium salt (WST-8) assay kit (CCK-8, Dojindo, Rockville, MD, USA) (Nojima et al, 2009). After cultivation for 7 d, adherent cells were detached with 0.25% trypsin and cultured with non-FBS EBM-2 in 96-well culture plates at 8×10 3 cells per well for 24 h. Then, the medium was replaced by fresh non-FBS EBM-2 with different concentrations of GB (0, 1.25, 5, 20, 80 and 160 μg/mL) for different periods.…”

“…EPCs are a type of cell population that have the capacity to circulate, proliferate and differentiate into mature endothelial cells, but have not yet acquired characteristic mature endothelial cell markers and have not yet formed a lumen (Asahara et al, 1997;Peichev et al, 2000).…”

DNA repair pathways in human multiple myeloma

“…Resistance to lMiDs involves mostly mutations or downregulation of cereblon expression. 47,137 Various mechanisms have been described for dexamethasone resistance: expression of a truncated glucocorticoid receptor, 138 mutations in receptor gene, 47 epigenetic silencing of RASD1, cell growth inhibitor induced by dexamethasone treatment, 139 overexpression of Hsp27, 140 or change in the secretion of cytokines and chemokines in the microenvironment. 141 Overexpression of Bruton tyrosine kinase could also play a role in resistance according to a recent study.…”