Methylation and gene silencing of the Ras-related GTPase gene in lung and breast cancers.
BACKGROUND
RRAD, a small Ras-related GTPase, is highly expressed in human skeletal muscle, lung, and heart. Although loss of expression of RRAD in breast cancer cells has been reported and it may act as an oncogene, the mechanism of silencing is unknown.


METHODS
We examined (1) mRNA expression of RRAD in lung and breast cancer cell lines using RT-PCR and (2) methylation status of lung and breast cancers.


RESULTS
Loss of RRAD expression was found in 14 of 20 (70%) NSCLC cell lines, 11 of 11 (100%) SCLC cell lines, and 8 of 10 (80%) breast cancer cell lines; expression was not affected in normal bronchial and mammary epithelial cells. Treatment of 23 expression-negative cell lines with a demethylating agent restored expression in all cases. We developed a methylation-specific assay from the analysis of bisulfite sequencing of the 5' region of RRAD in expression-negative and positive cell lines, which resulted in good concordance between methylation and expression. Primary lung and breast cancers showed hypermethylation in 89 of 214 (42%) and 39 of 63 (62%) cases, respectively. RRAD hypermethylation correlated with smoking history and poorer prognosis in lung adenocarcinomas.


CONCLUSIONS
We conclude that epigenetic silencing of RRAD is a frequent event in lung and breast cancers, and analysis of it may provide novel opportunities for prognosis and therapy of these cancers.
Suzuki
Makoto
M
Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan.
Shigematsu
Hisayuki
H
Shames
David S
DS
Sunaga
Noriaki
N
Takahashi
Takao
T
Shivapurkar
Narayan
N
Iizasa
Toshihiko
T
Minna
John D
JD
Fujisawa
Takehiko
T
Gazdar
Adi F
AF
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism; D018288:Carcinoma, Small Cell / Q000235:genetics / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C084355:RRAD protein, human; D018631:ras Proteins; D001374:Azacitidine