Genomic screening for genes silenced by DNA methylation revealed an association between RASD1 inactivation and dexamethasone resistance in multiple myeloma.
OBJECTIVE
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.


METHODS
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 RASD1 was 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.


CONCLUSIONS
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.
Nojima
Masanori
M
Departments of Public Health, SapporoMedicalUniversity, Japan.
Maruyama
Reo
R
Yasui
Hiroshi
H
Suzuki
Hiromu
H
Maruyama
Yumiko
Y
Tarasawa
Isao
I
Sasaki
Yasushi
Y
Asaoku
Hideki
H
Sakai
Hajime
H
Hayashi
Toshiaki
T
Mori
Mitsuru
M
Imai
Kohzoh
K
Tokino
Takashi
T
Ishida
Tadao
T
Toyota
Minoru
M
Shinomura
Yasuhisa
Y
D000970:Antineoplastic Agents / Q000627:therapeutic use; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D019175:DNA Methylation / Q000502:physiology*; D000077209:Decitabine; D003907:Dexamethasone / Q000627:therapeutic use*; D004353:Drug Evaluation, Preclinical; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004357:Drug Synergism; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing / Q000502:physiology; D005820:Genetic Testing; D006801:Humans; D008954:Models, Biological; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D018631:ras Proteins / Q000235:genetics* / Q000502:physiology
D000970:Antineoplastic Agents; C400132:RASD1 protein, human; D000077209:Decitabine; D003907:Dexamethasone; D018631:ras Proteins; D001374:Azacitidine